The sequence, structure, and modifications of tRNAs are exquisitely tuned for high fidelity charging and decoding, for efficient use in translation, and for high stability. One major goal of this project is to define the scope and specificity of a major quality control pathway in the yeast Saccharomyces cerevisiae that targets mature tRNAs lacking certain modifications for rapid tRNA decay (RTD), resulting in growth defects. We previously found that RTD acts on tRNAs lacking one or more of several modifications, is mediated by the 5'-3' exonucleases Rat1 and Xrn1, is inhibited by a met22? mutation, and acts on only a subset of tRNA species lacking the particular modifications. Understanding the specificity of this pathway is important because recent results in the field show that RTD is conserved in HeLa cells and that reduced tRNA levels occur in certain mouse modification mutants and in a number of mitochondrial diseases associated with tRNA mutations. Prior investigation of the tRNASer family showed that RTD occurs on fully modified and hypomodified tRNAs with destabilized acceptor and T-stems, resulting in increased exposure of the 5' ends, consistent with degradation by 5'-3' exonucleases. However, our recent high throughput analysis of tRNATyr function (using the SUP4oc nonsense suppressor) provides evidence that RTD is also triggered by a destabilized anticodon stem-loop (ASL), suggesting a new mechanism to engage RTD. In addition, we find evidence for an unexpected tRNA decay pathway that occurs on mature tRNAs in the absence of RTD. A second major project focuses on the biology of selected modifications associated with growth defects in yeast and with human disease. Defects in any of several different human modification genes are associated with intellectual disability, microcephaly, or familial dysautonomia. A major current goal is to understand the severe growth defect of cells lacking Trm7, which is required for 2'-O-methyation of C32 and N34 of 3 tRNA species. The human TRM7 homolog FTSJ1 is linked to non-syndromic X-linked intellectual disability (NSXLID). We found that the S. cerevisiae trm7? growth defect is due to lack of functional tRNAPhe and that tRNAPhe undergoes an intricate set of modifications in which Trm7 works with Trm732 and with Trm734 to catalyze Cm32 and Gm34 modifications, which then promote wybutosine (yW) formation at m1G37. We also found that this circuitry is retained and important in the distantly related yeast Schizosaccharomyces pombe. To follow up, we propose: (1) To define the mechanisms by which tRNA is recognized and degraded in yeast. (2) To define the mechanisms by which the RTD pathway is regulated, and (3) To determine the roles of selected modification genes implicated in human disease, with a focus on analysis of cell lines from NSXLID patients with FTSJ1 lesions, and on the roles and specificity of yeast Trm7 and its partner proteins.